Polytene Chromosome Map Research Articles

Nucleotide and protein distribution in BrdU‐labelled polytene chromosomes revealed by ion probe mass spectrometry

Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br- secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN- signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.

A cytogenetic analysis of the Simulium ochraceum species complex (Diptera: Simuliidae) in Central America

A cytobiotaxonomic study of the medically important insect vector Simulium ochraceum s.l. revealed two sibling species and one cytotype from various endemic and nonendemic zones of human onchocerciasis in Guatemala and Mexico. Polytene chromosome maps and idiograms as well as notes on the biology of the three taxa designated S. ochraceum A, S. ochraceum B, and S. ochraceum C within the subgenus Psilopelmia are presented. All three taxa exhibit distinct sex chromosomes and taxon-specific suites of autosomal inversion polymorphisms. Simulium ochraceum C differs from both S. ochraceum A and S. ochraceum B by five interspecific inversions designated IIS-7,8 and IIIL-12, 13 + 14, 15. The three taxa exhibit niche and biting preferences, with S. ochraceum A being highly anthropophilic. Analysis of autosomal inversion polymorphism profiles indicates S. ochraceum A has long-range dispersal capability. Our results are consistent with the general findings that in the Simuliidae, sibling speciation may be suspected wherever a morphospecies occupies different niches in a stream continuum. We find for the first time an apparent partitioning of taxa by altitude. Simulium ochraceum A may be a primary vector of human onchocerciasis in Guatemala, as its vertical distribution is coincident with that of the highest areas of nodule prevalence in the human population. A genotypic component of variation in vectorial capacity of S. ochraceum A populations seems to occur, since the Y4 chromosome and its X chromosome counterpart are associated with hyperendemic areas of human onchocerciasis. Our observation that a supernumerary band, 37B1Hb, is associated with sex determination in two of the taxa may be of significance for the elucidation of the molecular basis of sex determination and possible resolution of issues pertinent to the general model of sympatric speciation in the Simuliidae.

Localization of ribosomal DNA in Rhagoletis pomonella (Diptera: Tephritidae) by in situ hybridization.

The ribosomal DNA (rDNA) of Rhagoletis pomonella was localized within both salivary gland polytene chromosomes and somatic cell mitotic chromosomes by in situ hybridization using the heterospecific Drosophila melanogaster rDNA clone, Dm238. In situ hybridization analysis of polytene nuclei showed that R. pomonella rDNA is located in the nucleolus and adjacent granular network of chromosome 1. The site of origin of rDNA is within this isomorphic granular network. The preservation of nucleolar ultrastructure in some polytene chromosome preparations allowed light microscope localization of R. pomonella rDNA to the apparent periphery of fibrillar centres within fibrillar complexes. In somatic cell nuclei, DM238 hybridized to the nucleolus organizing region (NOR) located on chromosome 1 at the site of the secondary constriction. The frequency distribution of heteromorphisms for rDNA content, differential appearance of secondary constrictions, non-pairing of the NOR and differences in homologue lengths suggests that the structural differentiation of this region in chromosome 1 is sex linked. This the first published description of the salivary gland polytene chromosomes from R. pomonella, and we include a tentative karyotype description, polytene chromosome maps and comments on their suitability for banding and molecular analysis.

Genetic analysis of the foraging microregion of Drosophila melanogaster.

The rover/sitter polymorphism in Drosophila melanogaster larval behaviour is a unique example of a genetically determined, naturally occurring behavioural polymorphism. Allelic variation at the foraging locus (for) accounts for the rover (long foraging paths) and sitter (short foraging paths) phenotypes. We previously developed lethal tagging and used deficiency mapping to place for in the 24A3-C5 interval on the polytene chromosome map, thereby defining the for microregion. Here, we subjected this microregion to mutational analysis to (i) isolate putative lethal foraging mutations and characterize their behavioural phenotypes to assess whether or not for is a vital locus, (ii) generate cytologically detectable chromosome rearrangements with breakpoints in or near for for more precise localization and for future molecular analysis of the for gene, and (iii) identify other gene loci in the immediate vicinity of the for locus. We recovered 10 gamma-induced and 33 ethyl methanesulfonate (EMS) induced new mutations that define seven complementation groups in 24A3-D4. Two new EMS-induced lethal for alleles and four gamma-induced rearrangements with breakpoints in for were identified, which allowed us to further localize for to 24A3-5. All lethal mutations in for resulted in an altered behavioural phenotype providing evidence that both vital and behavioural functions are encoded by for.

Cytotaxonomy of the Simulium damnosum complex from central and northeastern Tanzania

A cytotaxonomic study of the medically important insect vector, Simulium damnosum s.l., revealed the presence of seven and possibly eight distinct taxa from central and northeastern Tanzania. Larval salivary gland polytene chromosome maps are presented for the first time for five cytotypes and one sibling species, which include the Nkusi form, the Sanje form, the Kisiwani form, Ketaketa C1 and C2, and the Kibwezi form. Inversion disequilibrium in males of the Kibwezi form indicate population substructuring is occurring and that this population may be in the early stages of speciation. Adults of the sibling species S. damnosum Kibwezi form and cytotype of the S. damnosum Nkusi form were identified using Malpighian tubule polytene chromosomes. The taxonomic status of the populations under study are discussed in relation to previously published papers and unpublished reports. Dimorphisms for centromere band enhancement occur on all three polytene chromosomes of the complement. The same centromere band can be polymorphic, sex linked, fixed, or lost in various cytotypes. In constructing a partial phylogeny, a hypothetical intermediate is proposed to account for the diverse fate of these centromere band dimorphisms and other inversion polymorphisms in different members of this nearly pan African complex. This pattern of chromosome restructuring is consistent with that seen for other species complexes within the Simuliidae.

Supernova (spno), a new maternal mutant producing variable-sized cleavage nuclei in Drosophila.

This paper describes a new recessive maternal lethal which disrupts normal nuclear division and migration during cleavage in Drosophila. We have named this gene locus supernova. Deletion mapping and in situ hybridization have located this gene to 88 F9/89 A1 on the polytene chromosome map. The terminal mutant phenotype is characterized by the presence of many variable-sized nuclei scattered throughout the cytoplasm of the unhatched egg. Following fertilization, the initial cleavage divisions appear delayed and are often accompanied by the formation of ring-like association of chromosomes and/or chromosome bridges. Although the polymerization of tubulin into spindles occurs during the initial cleavage divisions, there appears to be both a spatial and temporal uncoupling of DNA replication from the formation and proper functioning of spindles. Eventually no functional spindles are formed, but nuclei continue to increase in size and number with increasing age of the embryo following fertilization.

Zaprionus tuberculatus: chromosome map and gene mapping by DNA in situ hybridization.

The genus Drosophila has long been used as a model of karyotype evolution, demonstrating change by paracentric inversion and occasional centric fusion of an ancestral karyotype of five rod-shaped and one "dot" chromosome. This study shows, by mapping D. melanogaster probes hybridized to polytene chromosomes of Zaprionus tuberculatus, that this ancestral pattern extends beyond the genus Drosophila. A formal polytene chromosome map of Z. tuberculatus is presented.

Sequence, biochemical characterization, and developmental expression of a new member of the TGF-β superfamily in Drosophila melanogaster

More than 20 members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors have been implicated in development. One member of the TGF-β family has been previously reported from Drosophila, the decapentaplegic (dpp) gene which is involved in embryonic dorsal/ventral polarity, embryonic gut formation, and imaginal disk development. Using PCR methods, we have identified a second Drosophila gene in the TGF-β family. It encodes a protein product that is more similar to the TGF-β-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. Because of its localization on the polytene chromosome map, we refer to this gene as 60 A. Expression of a 60 A cDNA in Drosophila S2 cells was used to determine that 60 A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides. The carboxy-terminal peptides are recovered as disulfide-linked homodimers. The 60 A transcripts and protein are first detected at the onset of gastrulation, primarily in the mesoderm of the extending germ band. As the germ band retracts, and throughout later stages of embryonic development, the 60 A transcript and protein are most readily detected in cells of the developing foregut and hindgut.

Ovarian polytene chromosome map, notes on the status, morphology, biology and a new distribution record of Anopheles (Cellia) mousinhoi (Diptera: Culicidae)

Abstract. An ovarian polytene chromosome map of Anopheles mousinhoi is presented with a discussion of chromosome arm associations and X chromosome homologies with the An.marshallii complex of species. Scanning electron microscopy of the previously undescribed egg shows marked similarities between An.mousinhoi and An.funestus. Major morphological differences between An. mousinhoi and the An. marshallii complex are described. Anopheles mousinhoi is recorded from South Africa for the first time.

A Photographic Map of Drosophila madeirensis Polytene Chromosomes

A photographic map of salivary gland polytene chromosomes of Drosophila madeirensis has been constructed showing homologies and differences with respect to the standard gene arrangement of D. subobscura. Only two paracentric inversions in the X chromosome and some slight minor dissimilarities of one or two bands in the autosomes differentiate the chromosomes of these species.

The evolutionary history of D. buzzatii. XXII. Chromosomal and genic sterility in male hybrids of Drosophila buzzatii and Drosophila koepferae

The genetic basis of sterility in F1 male hybrids of Drosophila buzzatii and D. koepferae has been investigated in two steps. (1) By successive backcrossing of hybrid females to either parental species. (2) By assessment of the effects on male fertility of selected segments of polytene chromosomes from the donor species on a background entirely derived from the recipient species. The length of introgressed segments producing sterility was progressively reduced through repeated backcrosses. This procedure sometimes led to an approximate mapping of major genes of hybrid sterility (genic sterility) on the polytene chromosome map. At other times it was found that sterility was produced only when the introgressed segment exceeded a certain threshold size (chromosomal sterility). The contribution of the autosomes to hybrid sterility seems to be mainly of the chromosomal type. The evidence concerning the X chromosome is equivocal. No fertile males were found following introgression with any of the investigated segments of this chromosome. These results are compatible both with the presence of at least six major genes of hybrid sterility (genic sterility) and with the existence of a rather small threshold size for the chromosome segments producing sterility (chromosomal sterility). The role of the Y chromosome was not investigated in this study.

Polytene chromosome maps in the Medfly Ceratitis capitata

Polytene chromosome maps of the five autosomes from salivary gland cells in Ceratitis capitata are presented, and the more characteristic features of each element are described. The correlation of the polytene elements to miotic chromosomes and linkage groups is established by using various Y-autosome and autosome-autosome translocation lines. Two loci, dp (black pupal case) and w (white pupal case), are mapped to the third and fifth chromosome, respectively. In addition to the polytene maps presented, some extra figures of specific chromosomal regions are given for easier identification of each polytene element.Key words: polytene chromosome maps, Ceratitis capitata.

Genetic localization of foraging (for): a major gene for larval behavior in Drosophila melanogaster.

Localizing genes for quantitative traits by conventional recombination mapping is a formidable challenge because environmental variation, minor genes, and genetic markers have modifying effects on continuously varying phenotypes. We describe "lethal tagging," a method used in conjunction with deficiency mapping for localizing major genes associated with quantitative traits. Rover/sitter is a naturally occurring larval foraging polymorphism in Drosophila melanogaster which has a polygenic pattern of inheritance comprised of a single major gene (foraging) and minor modifier genes. We have successfully localized the lethal tagged foraging (for, 2-10) gene by deficiency mapping to 24A3-C5 on the polytene chromosome map.

Structure, frequency and distribution of P elements in relation to P—M hybrid dysgenic male recombination inDrosophila melanogaster

SummaryThe frequency and distribution of P elements were investigated in the third chromosomes of two wild-type strains ofDrosophila melanogasterusingin situhybridization of biotinylated probes to the polytene chromosomes. The relationship between these data and the extent of hybrid dysgenesis was determined through assays of egg production, egg hatchability (F2embryo lethality),snwdestabilization and male recombination along the third chromosome. The results suggest that P-element distribution, frequency and structure are all contributory factors in the regulation of hybrid dysgenesis. Texas 6 was shown consistently to be a stronger P strain than Texas 1, eliciting greater reductions in fertility, more extensivesnwdestabilization and higher frequencies of male recombination. Clustering of male recombination events, arising from pre-meiotic crossing over, was evident among the dysgenic progeny of each strain. Male recombination andsnwdestabilization were independently distributed among the dysgenic males studied, suggesting that these traits represent separate P-mediated functions. The third chromosome male recombination maps produced by the two strains differed significantly from each other and from the published female meiotic and polytene chromosome maps. Male recombination breakpoints were associated with the original distribution of P sequences in the two strains and the results suggest that this relationship may be closer for potentially complete P factors than for P sequences in general. An analysis of sub-lines derived from individual recombinant males revealed that chromosomal breakpoints could also be associated with novel insertions following P-element transposition.

Expression of a reporter gene resembles that of its neighbour: an insertion in the hairy gene of Drosophila.

Random insertions of a promotor fused to a reporter gene, such as Lac-Z, reveal regulatory sequences that confer temporal and spatial patterns of gene expression in eukaryotes. These patterns may reflect the activity of a neighbouring gene and thus lead to the isolation of new genes essential for normal development. Here, we demonstrate that this hypothesis is true for an insertion into the well characterized segmentation gene, hairy, in Drosophila. The insertion is homozygous lethal and fails to complement other hairy alleles, giving the phenotype described for hairy mutations. The insertion is located at 66D on the polytene chromosome map, is within 300-600 bp 5' to the first hairy exon, and is orientated in the same sense (5'-3') as the hairy transcription unit. Expression of β-galactosidase (β-gal), deriving from the insertion, follows closely the spatio-temporal patterns of expression of hairy gene product during embryogenesis. In addition, other sites of β-galactosidase expression are shown in the third larval instar stage and in the adult ovary. The results show that some insertions, giving restricted patterns of reporter gene expression, will reflect the temporo-spatial activity of a nearby gene.

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.

Polytene chromosome mapping in Ceratitis capitata (Diptera: Tephritidae)

Polytene chromosome reference maps of the five autosomes of Ceratitis capitata from male pupal orbital bristle trichogen cells are presented and a correlation is established between two of them and the two largest of the five autosomes in the haploid mitotic complement. Characteristic features of each chromosome are described identifying areas that are difficult to analyze and noting the existence of common alternative band expression. A quantitative analysis of the mitotic karyotype of C. capitata indicates that the two smallest autosome pairs cannot be reliably distinguished. This may present problems with future attempts to establish homologies between the remaining mitotic and polytene chromosomes. A comparison of polytene chromosome banding patterns from salivary gland and trichogen cells failed to find any homologous regions, or even to identify homologous chromosomes. The banding differences are not explained by variation in puffing patterns, heterochromatin expression, or polyteny levels, but appear to reflect fundamental differences in banding patterns of the chromosomes in each tissue. Key words: Ceratitis capitata, polytene chromosome map, mitotic chromosome measurements.

Enhancer of seizure: A New Genetic Locus in Drosophila melanogaster Defined by Interactions With Temperature-Sensitive Paralytic Mutations

Mutations in the enhancer of seizure (e(sei] locus have been isolated on the basis of their ability to cause temperature-induced paralysis of alleles at the seizure (sei) locus at temperatures at which these mutations ordinarily do not paralyze. This enhancer is specific to the seizure locus and is without effect on other temperature-sensitive paralytic mutants including para, nap, tip-E and shi. This suggests that the enhancer responds specifically to the mechanism of paralysis mediated by the seizure mutations. The e(sei) is a recessive mutation which maps to 39.0 on the left arm of chromosome 3. Deficiency mapping has placed it at 69A4-B5 on the salivary gland polytene chromosome map. When a new enhancer allele was isolated following P-M hybrid dysgenesis, there was a concomitant P-element insertion at 69B. In the absence of seizure mutations, the enhancer mutation causes non-temperature dependent hyperactivity when agitated and interferes with the climbing response. Electrophysiological studies examined the effects of increasing temperature on electrical activity in the adult giant fiber/flight muscle system. Neuronal hyperactivity was seen in both e(sei) and sei single mutant homozygotes, but not in wild type. The hyperactivity was more severe in the sei;e(sei) double mutants. The correlation between the physiological effects and the mutant behavior suggests that both sei and e(sei) cause membrane excitability defects. Since previous work has shown that seizure mutants affect [3H]saxitoxin binding to the voltage-sensitive sodium channel, e(sei) may code for a gene product which interacts with this channel.

Sequence and genomic structure of ras homologues Dm ras85D and Dm ras64B of Drosophila melanogaster

The ras homologues of Drosophila melanogaster located at 85D and 64B on the polytene chromosome map were cloned using the Ha- ras gene of Harvey murine sarcoma virus as a probe. The genomic sequences of Dm ras85D and Dm ras64B were determined and shown to differ from previously published sequences. Dm ras85D is much more similar to the Ha- ras, Ki- ras, and N- ras genes than it is to either the Dm ras64B gene or to the ras genes of Saccharomyces cerevisiae. Comparison of the Dm ras85D genomic sequences with the previously published nucleotide sequence (Neuman-Silberberg et al., Cell 37 (1984) 1027–1033) shows that the positions of the two introns are not conserved relative to the positions of the introns in Dm ras64B or in vertebrate ras genes. The Dm ras64B and Dm ras85D transcripts were analyzed by blot hybridization and shown to be dissimilar. The data suggest that the divergence of the Dm ras genes was ancient, and that Dm ras85D and Dm ras64B have different functions.

A complete and a truncated U1 snRNA gene of Drosophila melanogaster are found as inverted repeats at region 82E of the polytene chromosomes.

A phage containing two sequences homologous to U1 snRNA was isolated from a Drosophila melanogaster genomic library, and identified with a previously cloned D. melanogaster U1 snRNA gene. DNA sequence analysis showed that complete and truncated U1 snRNA genes are present, both of which have base substitutions relative to U1 snRNA. These genes show conservation of 5' and 3' flanking regions relative to other U1 and U2 snRNA genes of Drosophila. Intramolecular renaturation experiments and electron microscope mapping demonstrates that the two U1 snRNA sequences are present as inverted repeats about 2.7kb apart, separated by a smaller pair of inverted repeats of an unrelated sequence. These U1 snRNA sequences were located by in situ hybridization at 82E, and related sequences were found at 21D and 95C on the polytene chromosome map. The results are discussed with reference to the origin and function of snRNAs.